Abstract
Incidental to the observations that a close association exists between tetanospasmin and hemoglobin in the blood of guinea pigs suffering from tetanus intoxication 1 a series of experiments was carried out on combination of hemoglobin with bacterial toxins in vitro. The results are briefly presented in this communication.
The toxins employed were filtrates from the “agar washings” of cultures of meningococcus and B. typhosus which were highly potent in the elicitation of the phenomenon of local skin reactivity. 2 In this work they were purified by dialysis in cellophane bags No. 600 against 0.85% NaCl solution for a period of one week. 3
The hemoglobin preparations were made in the following manner: All the work was done under strict precautions of sterility and each step controlled for bacterial contamination on aerobic and anaerobic media. Rabbit blood obtained from the heart was defibrinated by shaking with glass beads, filtered through several layers of gauze and centrifuged in order to separate the plasma from the erythrocytes. The erythrocytes were promptly washed in 1 % NaCl solution by repeated centrifugalization until the washings became biuret-negative (from 5-7 washings being required). The packed red blood cells were kept frozen for several hours at -70°C in a mixture of cellosolve and dry ice and gradually thawed out in a mixture of ice and alcohol and in the refrigerator at 4°C overnight. The cells were diluted in distilled water to a hemoglobin concentration of 50-60% as determined in the Hellige haemometer.
Filtration of the solution through a Seitz filter resulted in effective removal of cellular debris, as ascertained by examination of spreads stained by Wright's method and hanging-drop preparations. In control experiments adjusting the pH to 5.5 by the addition of N/10 HC1 gave no precipitate in the filtrates. 4
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